The invention relates to methods for the purification of biological solutions by removal of endotoxin, including the ex-vivo depletion of endotoxin from blood.
Bacterial endotoxins are lipopolysaccharides (LPS) derived from the outer cell membranes of Gram-positive and Gram-negative bacteria (Vaara and Nikaido, 1984). Endotoxins are known to have potent biological effects in human. Serious endotoxin infection can cause sepsis and septic shock, leading to severe hypertension, cardiovascular collapse, multiple organ failure and death (Billiau and Vandekerckhove, 1991). In biotechnology industry, bacteria are widely used to produce recombinant DNA products such as peptides and proteins. Bacterial endotoxins have been recognized by the industry as a major cause of the pyrogenic reactions that can be encountered during the administration of biotherapeutics (Hou and Zaniewski, 1990). The removal of these physiologically active agents from final bioproducts has always been a challenge, especially in the situations where endotoxins bind product proteins. Significant product loss and low product yield can result from the separation steps employed to remove endotoxins.
Although numerous methods such as ion exchange adsorption (Webber et al., 1995), ion exchange membrane (Belanich et al., 1996), ion-exchange filter (Hou and Zaniewski, 1990), ultrafiltration (Li and Luo, 1998, 1999), extraction (Aida and Pabst, 1990) have been studied for endotoxin removal from protein solutions, affinity adsorption has proven to be the most effective technique. Polymyxin B has been investigated intensively as an endotoxin binding ligand since Issekutz (1983) reported its ability to remove endotoxin from solutions. In subsequent studies, polymyxin B has been immobilized on chromatographic supports such as Sepharose (Karprus et al., 1987) to remove endotoxin from protein solutions. Membrane (Petsch et al., 1997) or fiber (Tani et al., 1992) have been used as the support as well. Immobilized histidine has also been employed to remove endotoxin from protein solutions. Sepharose resin (Matsumae et al., 1990), filter-paper (Guo et al., 1997), and hollow fiber membrane (Legallais et al., 1997) have been used as the support. Hirayama et al. (1994) studied the use of cross-linked N,N-dimethylaminopropylacrylamide (DMAPAA) spherical particles for selective removal of endotoxin from protein solutions. They found that ionic strength had less effect on the endotoxin removal efficiency with DMAPAA than with immobilized histidine. An ACTICLEAN ETOX affinity column from Sterogene Bioseparations (Carlsbad, Calif.) has been used to remove endotoxin from hemoglobin preparations where the protein formed complex with LPS subunits (Kang and Luo, 1998). The effects of various solutions such as endotoxin-free water, NaCl and CaCl2 on the endotoxin removal efficiency and protein recovery in the chromatographic process were investigated.
Immobilized metal affinity chromatography (IMAC, also know as metal chelate chromatography) was introduced as a new technique for protein purification by Porath et al. in 1975. Since then IMAC has gained wide acceptance in purification of proteins and peptides (Sulkowski, 1985; Yip and Hutchens, 1994; Scopes, 1994). Much of the earlier work concentrated on using Cu2xe2x88x92 or Zn2+ as the chelating metal ion (Sulkowski, 1985; Scopes, 1987). Later Fe3+ was found to be a very effective chelating ion for isolation of phosphorylated amino acids, peptides and proteins based on the preferential affinity between the immobilized iron (III) ion and the phosphate groups in the biomolecules (Andersson and Porath, 1986; Muszynska et al., 1992).
Numerous methods and devices have been described for the extracorporeal (ex vivo) removal or treatment of various blood components by circulating blood outside of the body through an apparatus containing membranes or particulate supports to which are attached binding agents for the component to be removed. For example, heparinase has been attached to a particulate support to degrade heparin in blood U.S. Pat. No. 4,373,023); chelants to remove metal ion oxidants have been described for the treatment of atherosclerosis (U.S. Pat. No. 5,753,227); and an adsorbent for removing low density lipoprotein (LDL) and endotoxins (U.S. Pat. No. 5,476,715), the endotoxin bound using a homo-, co-, or terpolymer of acrylic acid and/or methacrylic acid.
It is towards the development of improved methods for depleting endotoxin from biological and other solutions that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
In its broadest aspect, the present invention is directed to a method for depleting endotoxin from a solution comprising the steps of: (1) providing an immobilized metal affinity chromatography matrix comprising a metal ion; (2) exposing the solution to the matrix under conditions wherein the endotoxin contained therein binds to the matrix; and (3) collecting the solution after exposure to the matrix, wherein the solution is depleted of endotoxin. Various adsorbent materials or matrices may be used for the aforementioned purpose, in the form of beads, fibers, or other formats, comprising, by way of non-limiting example, various plastic resins such as polystyrene, polymers such as poly(hydroxymethacrylate), agarose, and the like. The metal binding or chelating moiety of the matrix may be, for example, iminodiacetic acid, nitrilotriacetic acid or tris(carboxymethyl)ethylenediamine. These moieties bind a metal ligand. Metal ions (metal ligands) useful for this purpose include but are not limited to iron (III), copper (II), cobalt (II), nickel (II), zinc (II), cerium (III), magnesium (II), calcium (II), chromium (III), lanthanum (III), lutetium (III), scandium (III), thallium (III), ytterbium (III), thorium (IV), and uranate (II). Iron (III) is preferred.
In one embodiment of the invention, in step (3) above the matrix is exposed to an elution buffer to elute material other than endotoxin that has bound to the matrix. The elution buffer may be by way of a non-limiting example, a phosphate buffer, and may be present at a concentration of about 0.02 M to about 0.06 M. In another embodiment of the invention, the matrix is provided in the form of a column, the solution to be depleted of endotoxin is passed through said column, and subsequently the column is washed with a buffer to collect material not adhered to said matrix. Proteins that may adhere to the matrix may be eluted with an elution buffer which displaces the proteins but does not release endotoxin. The elution buffer may be, for example, a phosphate buffer, and may be present at a concentration of about 0.02 M to about 0.06 M. In yet a further embodiment, the solution to be depleted of endotoxin is adjusted such that upon exposure to the aforementioned matrix, endotoxin is selectively bound to the matrix but proteins are not. Such adjustment may be provided using phosphate, such as a phosphate buffer, in the solution. A concentration of phosphate of about 0.02 M to about 0.06 M achieves this purpose, although this may be varied within the scope of the invention. In a further example of the practice of the invention, the matrix may be regenerated after use by treatment with a solution of a metal chelator to remove the metal and endotoxin bound thereto, followed by washing and recharging with metal ion. A non-limiting of a metal chelator useful for this purpose may include but is not limited to ethylenediaminetetraacetic acid (EDTA); ethylene glycol-bis(beta-aminoethyl ether)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA); ethylenediamine-N,Nxe2x80x2-diacetic acid (EDDA); or nitrilotriacetic acid (NTA).
In another broad aspect of the invention, a method is provided for the extracorporeal depletion of endotoxin from the circulation in a patient. In this method, the patient""s blood is exposed ex vivo to an immobilized metal affinity chromatography matrix comprising a metal ion as described hereinabove, wherein endotoxin in the blood is bound by the matrix, and the blood is subsequently returned to the patient""s circulation.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.